Project 3 addresses the Center for Tobacco Products (CTP) research priorities under Health Effects scientific domain as defined in RFA-OD-17-006, and the overarching integrative scientific theme of this TCORS coalesce for testing the effects of flavorings in tobacco products and characterize their respiratory toxicity. Many of the flavorings chemicals being used in emerging tobacco products, including electronic nicotine delivery system (ENDS), waterpipe tobacco and cigarillos, were evaluated for safety through ingestion only. These flavorings have not been tested adequately for safety when inhaled in aerosolized form. At present, no regulatory program exists to assess the respiratory health hazards of the flavorings in tobacco products. This project will focus primarily on respiratory health effects, including inflammation, biomarkers of exposure, and clinical markers with relevance to disease endpoints that will directly supports the Toxicity scientific domain. Embedded within these health effects studies, we will of necessity gather data on product usage patterns that may influence health effects, including total consumption, topography, and flavor preference, which will help inform the Behavior scientific domain. We propose to test the hypotheses that inhalation of flavored ENDS aerosol has the potential to cause chronic respiratory tract inflammation, and that potential inflammatory effects and observed respiratory side-effects associated with flavored ENDS use will depend on the specific flavoring chemicals used in the products and the patterns of products use. Our goal is to better characterize the respiratory health effects attributable to the use of flavored tobacco products. We propose two human studies to evaluate respiratory health effects from inhaling flavored products among daily ENDS users as they switch among various flavors. These two studies aim to: AIM 1 (Study 1): Assess changes in respiratory symptoms and biomarker levels among ENDS users during spontaneous `switching' between flavors; AIM 2 (Study 2): Evaluate respiratory symptoms in ENDS users switching from flavors of potentially high-toxicity to flavors of potentially low-toxicity; and AIM 3 (Studies 1&2): Characterize ENDS users' preferences, selection, patterns of use and switching between various flavors. In Study 1, we will establish a cohort of ENDS users to assess changes in respiratory symptoms during spontaneous `switching' among flavors over one year. In Study 2, we will conduct a randomized, parallel-group open-label trial to evaluate respiratory symptoms in ENDS users switching from higher-toxicity to lower-toxicity flavors (as identified by Project 1). Endpoints include clinically relevant biomarkers of inflammation and oxidative stress, changes in respiratory function, subjective respiratory symptoms and side-effects, and expression of immune and inflammatory response genes in nasal epithelial cells. Outcomes for Regulatory Science: Assessment of respiratory health effects in ENDS users will provide crucial information related to safety of flavored tobacco products, which would inform the FDA's regulatory efforts, including product standards.